ABSTRACT Heterosexual transmission of HIV-1 remains the dominant mechanism by which the epidemic is sustained worldwide and increasingly is the route by which the virus is spreading within the US. It is now well established that in a majority of transmissions, infection is initiated by a single genetic variant, the transmitted founder (TF) virus, from the transmitting partner quasispecies. This severe severe genetic bottleneck, in which one or a limited number of variants from the diverse population present in the transmitting (donor) partner establish productive infection in the previously uninfected partner is the result of both stochastic and selective processes. Although we and others have shown that selection for less evolved (more ancestral) variants present in the donor?s viral quasispecies are favored during selection, the biological traits that define this enhanced transmissibility remained to be fully elucidated. In this renewal application, we are proposing 3 Specific Aims that combine cohort-level viral genetics analyses, in vitro phenotypic characterization, as well as an in vivo model for HIV-1 transmission, to define the viral properties that are advantageous during transmission. These approaches together have the potential to provide more definitive information on whether there are biological phenotypes of HIV-1 that are linked to transmission and could be targeted for intervention. Specifically we will: 1. Define specific virologic traits that correlate with the selection of more consensus-like viruses through a broad analysis of subtype A and C transmission pairs, coupled with novel assay methodologies. 2. Characterize the nature, impact and extent of selection bias across genomic regions and subtypes using a novel PacBio sequencing approach to define the donor quasispecies and the TF virus. 3. Determine, using humanized BLT mice, whether preferential transmission of TF viruses over donor- matched NT viruses occurs in this model and reveals viral and host determinants of transmission. A knowledge gap remains in our understanding of the genetic bottleneck associated with HIV-1 transmission. The experiments proposed will narrow this gap by conducting a thorough interrogation of the biological characteristics of the transmitted founder virus that are linked to its preferential transmission, using an expanded panel of transmission pairs from two African cohorts and novel technologies that have not been applied in this setting. Such traits, if defined, could provide new avenues for specifically targeting prevention approaches to the viruses most likely to initiate new infections.